Calcium is a key element in the regulation of numerous cellular processes. For example, the development of tension in vascular smooth muscle and cardiac muscle is dependent on a rise in free cystolic Ca.sup.++ levels. Also, it is known that changes in levels of intracellular Ca.sup.++ are linked to physiological events as diverse as platelet aggregation exocytosis and cell proliferation.
The role of Ca.sup.++ in important cellular processes has directed investigation into the development of techniques for measuring calcium ion levels in living cells. The use of optical indicators, in particular tetracarboxylate compounds, appears to be the most reliable method of calcium ion detection. The tetracarboxylate compounds in the form of an ester derivative penetrate the cell membrane and are then enzymatically cleaved in the cell to give tetracarboxylate ions which are impermeable (or permeable only at a slow rate) to the cell membrane. When the tetracarboxylate compounds bind to calcium ion, a shift in the ultraviolet spectrum is produced. This shift is used to measure the amount of bound calcium ion and from a knowledge of the dissociation constants, the intracellular calcium concentration can be determined.
In 1985, the following two tetracarboxylate chelators for calcium detection were reported (Grynkiewicz et al, J. Biol Chem. 260:3440) as improved Ca.sup.++ indicators: ##STR1##
However, the use of Fura-2 and Indo-1 in flow cytometry, which is a very efficient method for measurement of parameters on individual cells (Muirhead, Trend. Anal. Chem. 3:107, 1984), is severely limited by their short absorption wavelength which required use of high power lasers with ultraviolet capabilities. The compounds of the present invention have longer absorption wavelength characteristics which make them useful for multiparameter flow cytometric analysis of intracellular calcium ion concentrations in mixed cell populations.